JP4558947B2 - Kit for labeling proteins with yttrium-90 - Google Patents

Abstract

Methods and kits for radiolabeling proteins and peptides with radiolytic isotopes, particularly yttrium-90, are disclosed, whereby sufficient purity, specific activity and binding affinity are achieved such that the radiolabeled protein may be directly administered to a patient without further column purification. Such kits and methods will be particularly useful in bringing radioimmunotherapy to the hospital and outpatient setting for the treatment of cancer.

Description

調製した6個の確認ロットについて、結合が得られた割合は、78.6％〜87.0％の範囲であり、平均が82.3％ある。Y2B8の放射線取り込み値の平均は、98.8％（96.3％〜99.5％の範囲）である。全体として、これらの結果からは、放射性同位体ラベルキット法によるY2B8の調製の再現性、および困難性が確認され、全体として、この放射ラベルキットを使用して調製されたY2B8は、臨床設定における使用に適していることを示している。
【００７３】
例2. 反応パラメーターの初期評価〜pHおよび反応時間
様々なpHおよび反応時間の条件下でラベル反応を行った後、初期段階の速度実験を行って、⁹⁰Yラベルされた抗体の放射取り込み、および結合を評価した。pH3.9から4.7の範囲で、5分間インキュベートして放射ラベル反応をした場合、＞80％がCD20陽性細胞との結合を維持したままで、＞96％の放射取り込みであった（表2）。pH2.9から4.6の範囲で、3、5、および10分間インキュベートした場合にも同様の結果が得られた（表3）。
【００７４】
【表２】

【００７５】
【表３】

Y2B8プレパレーションの免疫反応性は、Lindmo et alの方法を使用して測定した。新たに回収したCD20陽性SB細胞の量を増やして、抗原過剰の条件下で、固定した量のY2B8とインキュベートした。逆数プロットによる結合データの解析では、一回の調製の後に、Y2B8の免疫反応性は72.2％を示した（図1）。
【００７６】
例3. さらなる反応パラメーターの評価
I 導入
この節において記載された実験は、Y2B8放射ラベルキットを使用して調製したY2B8の結合について、プロトコールのずれに対する影響を調査した。放射ラベルされた抗体の結合は、放射ラベルの工程におけるいくつかのパラメータの影響を受けるであろう（表4）。
【００７７】
【表４】

放射ラベルプロトコルに由来する以下のずれは、結合に対して最も負の影響を有するであろうものとして同定され、かつ含んでいた：
1.）より少ない量の酢酸ナトリウムの添加、
2.）過剰量の塩化物溶液の添加、
3.）より少ない量の2B8−MX−DTPAの添加、および、
4.）最大反応時間を上回るインキュベーション、
である。これらのずれの影響は、別々に、または同時に評価した。
【００７８】
別々に評価した場合、8分間インキュベートしたときでさえ、上記項目の1−3における20％量のずれでは、IDEC−Y2B8が、臨床試験において結合のために確立された放出規格に合格する結果となった。
【００７９】
三つの量全てのずれを同時に起こした研究において、月曜日ラベルプロトコル（放射線分解が最大の可能性がある）を使用して調製され、および8分間（通常より60％長い）培養した用量の場合にのみ、臨床放出規格よりわずかに下であった（＜3％）。逆に、金曜日ラベルプロトコルを使用して調整された用量では、4種のパラメーターの全てにおいて（上記1−4）ずれの累積的効果にも関わらず、許容可能な結合結果を維持した。全てのずれにおいて別々および同時起こる場合、放射取り込みが、臨床放出規格の95％より上であった。
【００８０】
II. パラメータの選択
我々は、必要とされる試薬量の20％のずれ、または通常使用される最大6分の反応時間の30％過剰までの許容が、放射薬学で使用されるプロトコルからの最大限可能なすれを示すと判断した。本研究において、我々は、IDEC−Y2B8の結合に対するこれらのずれの影響を評価した。我々は評価した条件が、週の全体にわたる用量調製の極限値を示すことを立証するために、「月曜日」および「金曜日」ラベルをシミュレートした。全てのずれが単一の用量調製に生じた場合の結合に対する組合わせの効果、および⁹⁰Yの放射取り込みに対する、これらのずれの影響を評価した。
【００８１】
「月曜日」および「金曜日」ラベルは、次の考えを反映している。⁹⁰Y塩化物溶液が短い半減期（64h）を有するので、使用した放射性同位体の量は、用量を調製した週のうちの日にちに依存する。このような理由により、月曜日に用量調製した反応量はより少なく、その結果⁹⁰Yの濃度がより高くなり、おそらくより強い放射線分解を生じる。従って、我々は、月曜日および金曜日ラベル法をシミュレートして、週の全体において用量調製の極限値を示すことを立証した。
【００８２】
III. 材料および方法
A. 試薬
1. 0.05M HCl中の⁹⁰YCl；Pacific Northwest National Laboratory, reagent grade; P.O.#08016,08118、
2. Ultrex HAL; J.T. Baker, Product#6900, Lot#J22539、
3. 洗浄用の滅菌水；Baxter, Part#2F7114, Lot#G926092、
4. 希釈バッファー；10mM PBSを含むpH7.4、1％BSA；Sigma, Part#P-3688, Lot#076H8913、
5. IDEC Supplied Radiolabeling kit;IDEC Part#130018, Lot#0129、以下のものを含む：
a.)2B8−MX−DTPA；IDECPart#129017, Lot#0165、
b.)50mM 酢酸ナトリウム；IDEL Part#121017, Lot＃0209A、
c.)調合バッファー；IDEC Part#120015, Lot#0202、
d.)反応バイアル；IDEC Part#122015, Lot#0218、
6. 凍結乾燥SB細胞、IDEC Part#127, Lot#127-001F、
B. 材料および装置。
【００８３】
1.ピペッター（20，200、および1000gL）、
2. ボルテックス、
3. 金属フリーピペットチップ（Biorad; metal-free）、
4. ガンマカウンター（Isodata, Model#20-10）、
5. ガラスチューブ（12×75mm）、
6. ポリプロピレンチューブ(Costar; 15mL および50mL conical, 滅菌)、
7. Tec-Control Radiochromatographic Kit（BioDex；Cat＃151-770）、
8. マイクロ遠心機（Savant）、
9. ポリプロピレンマイクロフージチューブ、金属フリー（Biorad;Cat#223-9780）。
【００８４】
C. 方法
1. Y2B8の調製
一般的に、 ⁹⁰Yラベルされた2B8−MX−DTPAは、以下に記載した変更により修飾された、上記の小スケールバージョンの放射ラベルキットプロトコルを使用して調製した。放射ラベルは、保存濃度が84mCi/mLまたは29.8mCi/mLの濃度の⁹⁰Y塩化物を使用して行い、それぞれ月曜日または金曜日用量のプレパレーションをシミュレートした（水曜日キャリブレーションである50mCi/mLを基にして）。濃縮された⁹⁰Y塩化物溶液は、50mM HCl(Ultrex, high-purity)を使用して、「金属フリー」のプラスチックマイクロフージチューブ内で希釈した。Ultrex(high-purity)HClを洗浄用の滅菌水（SWFI）で50mMに希釈した。放射ラベル反応は、「金属フリー」のプラスチックマイクロフージチューブ、15mlのコニカルチューブ、またはY2B8放射ラベルキットにより提供された10mLのガラス中隔反応バイアル内で行った。
【００８５】
a. 小スケールラベルから全スケール用量の調整
0、3、10、および40mCiの放射ラベル反応は、月曜日用量プレパレーションをシミュレートする反応条件を使用して行った。各反応の試薬量をmLsとして表5に抄録した。
【００８６】
【表５】

5分間インキュベートした後、20μlサンプルを除去して、調合バッファーで最終抗体濃度を0.21mg/mLに希釈して、アッセイするまで2-8℃に保存した。このレポートに記載された以下の全ての実験において、コントロールとして1mCi反応液を使用したので、結合値を1mCi反応液に標準化した。報告された値は、コントロールの結合値により各反応の結合値を割ることによって1mCiコントロールサンプルとして標準化し、割合として表示した。
【００８７】
b. 添加した酢酸ナトリウムの量に対する影響
月曜日ラベルにおいて、10mCiの⁹⁰Y塩化物（0.119mL）と0.114mLの50mM酢酸ナトリウムを混合した。この50mM酢酸ナトリウムの量は、IDEC−Y2B8の臨床において用量を調整するために通常使用されるバッファーの量よりも20％少ない。結合した抗体（2B8−MX−DTPA）を添加して（0.333ml）、前記サンプルを混合して、次に周囲温度でインキュベートした。放射ラベル溶液の特異的活性は18.9mCi/mg抗体であった。2分の時点で、0.020mLを除去して、調合バッファーで0.24mg/mLに調合して、2-8℃に保存した。8分後、放射ラベル溶液の残りを0.24mg/mLに調合して、2-8℃に保存した。金曜日ラベルをシミュレートするために、0.336mlの⁹⁰Y塩化物、0.323mLの酢酸ナトリウム、および0.333mlの2B8−MX−DTPAを使用して、前記プロトコルを繰り返した。両研究において、上記の「標準」条件を使用して、1mCiコントロース反応を行った（5分反応）。
【００８８】
c.添加した⁹⁰Y塩化物の量に対する影響
月曜日ラベルにおいて、12mCiの⁹⁰Y塩化物（0.143mL）を0.143mLの50mM酢酸ナトリウムと混合した。この⁹⁰Yの量は、一般的にY2B8を用量調整するために通常使用される⁹⁰Yの量よりも20％多い。結合した抗体を添加して、前記サンプル溶液を混合して、次に周囲温度でインキュベートした。最終的な放射ラベル溶液の特的活性は22.5mCi/mg抗体であった。2分の時点で、0.020mLを除去して、調合バッファーで0.24mg/mLに調合して、2-8℃に保存した。8分後、放射ラベル溶液の残りを0.24mg/mLに調合して、2-8℃に保存した。金曜日ラベルも同様に、0.403mlの⁹⁰Y塩化物、0.403mLの酢酸ナトリウム、および0.333mlの2B8−MX−DTPAを使用して実施した（特異的活性は22.5mCi/mg抗体であった）。両研究において、上記の「標準」条件を使用して、1mCiコントロース反応を行った（5分反応）。
【００８９】
d.抗体結合体の量を少なく添加したときの影響
月曜日ラベルにおいて、10mCiの⁹⁰Y塩化物（0.119mL）と0.143mLの50mM酢酸ナトリウムを混合した。通常使用される抗体の量よりも20％少ない結合した抗体を添加して（0.267ml）、前記サンプルを混合して、次に周囲温度でインキュベートした。2および8分の時点で、0.020mLを除去して、調合バッファーで最終抗体濃度を0.24mg/mLに調合して、アッセイまで2-8℃に保存した。金曜日ラベルも同様に、0.336mlの⁹⁰Y塩化物、0.403mLの酢酸ナトリウム、および0.27mlの結合体を使用して、実施した。両研究において、上記の「標準」条件を使用して、1mCiコントロース反応を行った（5分反応）。
【００９０】
e.試薬のずれを組み合わせたときの影響
月曜日または金曜日ラベルプロトコルにおいて、酢酸ナトリウム、⁹⁰Y塩化物、および結合体の量を20％すらしたときの影響を同時に評価した。月曜日ラベルにおいて、12mCiの⁹⁰Y塩化物（0.143mL）を0.113mLの50mM酢酸ナトリウム、20％多い量を示す⁹⁰Y塩化物、通常使用される酢酸ナトリウムの20％少ない量と混合した。通常使用される抗体よりも20％少ない量を示す2B8−MX−DTPAを添加して、前記サンプル溶液を混合して、その後周囲温度でインキュベートした。2、4、6および8分の時点で、0.020mLを除去して、調合バッファーで最終抗体濃度を0.24mg/mLに調合して、アッセイまで2-8℃に保存した。金曜日ラベルも同様に、0.403mlの⁹⁰Y塩化物、0.387mLの酢酸ナトリウム、および0.267mlの結合体を使用して実施した；40μLのサンプルを示した時間に除去して、調合バッファーで調合した。両研究において、上記の「標準」条件を使用して、1mCiコントロース反応を行った（5分反応）。
【００９１】
2. 放射取り込みの測定
上記アッセイに従って、Biodex（Tec-Control Radiochromatographic Kit）によって製造された商業的に利用可能なキットを使用して、結合体と結合した放射活性の量を測定した。一般的なマイクロピペッターを使用して、0.5-1μlのサンプルを複製ストリップに注ぎ、かつBiotex instruction incertによって展開した。Isodataガンマカウンターを100-1000KeVのウィンドーで使用して、ガラスチューブ内でストリップの半分の放射活性を計測した。放射ラベルの取り込みは、上部および底部の半分の両方から求めた全活性から前記ストリップの上部半分の放射活性の量をわけることによって評価した。この値は、パーセントとして、かつ測定した平均値を示した。
【００９２】
3.結合の測定
上記プロトコルに従って、サンプルのCD20陽性細胞に対する結合％を解析した。しかし、ネガティブコントロールであるHSB細胞のサンプルは、これらの実験には含まれておらず、およびSB細胞はマイクロフージチューブの代わりに5mlバイアル内で凍結乾燥した。
【００９３】
本質的に、最終的に調合した全てのY2B8サンプルは、希釈バッファー（10.0μlの抗体+990μLバッファー）で1：100に希釈した。次に前記抗体は、50mLポリプロピレンチューブ内の10mlの希釈バッファーに、30μLの1：100希釈液を添加することによって8ng/mLの範囲の濃度に再び希釈した。
【００９４】
6から7個の凍結乾燥細胞のバイアルをSWFIで元に戻して、50mLコニカルチューブにプールした。再構成された細胞（0.5mL）を三本の1.5mLマイクロフージチューブ内に三等分して、サンプルあたり三本をテストした。希釈バッファー（0.5mL）を三本の空のマイクロフージチューブに添加した。希釈した抗体（0.5mL）をそれぞれのチューブに添加して、しっかりとふたをして、周囲温度で45分間、エンドオーバーエンドで混合してインキュベートした。インキュベーションした後、「6」（4000×ｇ）にセットしたSavant マイクロ遠心機を使用して、5分間遠心することによって細胞をペレットとした。エネルギーウィンドーを100-1000KeVにセットしたIsodata ガンマカウンターを使用して放射活性を測定するために、前記サンプルの上清（0.75mL）を12×75mmのガラスチューブに移した。
【００９５】
細胞に結合した放射活性（B）は、添加した全放射活性から未結合（上清）の放射活性を引くことによって見積もった。全放射活性は、細胞のないチューブの放射活性を計測することによって測定した。結合％は、結合した放射活性を全パーセントとして示すことによって見積もった。
【００９６】
結合を評価するために使用する凍結乾燥された細胞間のロットのばらつき効果を最小にするために、「標準的」ラベル条件を使用して調製された1mCiのY2B8コントロールによって結合値を標準化した。この節の始めの方に述べたように、各セットの実験に対してコントロールサンプルを調製した。
【００９７】
D.結果
1.全スケールでの用量のプレパレーションを予測するための小スケールラベル 小スケール放射ラベル反応から、全スケール（40mCi）用量のプレパレーションが予測されることを確認するために、上述の放射ラベルプロトコルを使用して、1、3、10、および40mCiのY2B8用量を調製した。これらの結果を表6に示し、および反応混合液のスケールを1ｍCi〜40mCiに増加しても、結合、または放射取り込みに対して不利な影響がないことが示される。
【００９８】
【表６】

2.酢酸ナトリウムの量を少なく添加した場合の影響
20％少ない量の酢酸ナトリウムを使用して、かつインキュベーション時間を60％のばしてY2B8を調製した場合、以下の「標準的」ラベル条件で調製された放射ラベルと比較して、実質的な結合が保持された（以下の表7）。ラベル反応が月曜日ラベル条件を使用して実施された場合でさえ、コントロール結合の＞89％が保持された。同様の結果が、金曜日用量のプレパレーションからも得られた。このようなずれは、用量を調製した日にちにかかわらず、放射取り込みに影響を与えなかった。
【００９９】
3.⁹⁰Y塩化物の量を過剰に添加した場合の影響
20％過剰量の⁹⁰Y塩化物を使用して、インキュベーション時間を通常使用されるものよりも60％長くすることと組み合わせて、Y2B8を調製した場合、以下の「標準的」ラベル条件によって調製された放射ラベルと比較した場合の、実質的な結合が保持された（以下の表7）。ラベル反応を8分間増加した場合、月曜日または金曜日用量プレパレーションのいずれにおいても、コントロールと比較して、結合は＞90％のままであった。20％多い量の⁹⁰Y塩化物を添加しても、用量を調製した日にちにかかわらず、放射取り込みに影響を与えなかった。
【０１００】
4.抗体結合体の量を少なく添加した場合の影響
20％少ない量の結合体（2B8−MX−DTPA）を使用して、かつインキュベーション時間を60％のばしてY2B8を調製した場合、以下の「標準的」ラベル条件で調製された放射ラベルと比較して、結合は有意な影響を受けなかった（以下の表7）。20％少ない量の結合体を添加しても、用量を調製した日にちにかかわらず、放射取り込みに影響を与えなかった。
【０１０１】
【表７】

5.試薬のずれと組み合わせた場合の影響
四種のずれの全てを同時に起こしたプロトコルを使用してY2B8を調製した場合、「標準的」ラベル条件で調製された放射ラベル抗体と比較して、結合はいまだ実質的に維持された（以下の表8）。月曜日プレパレーションを通常使用される最大の6分よりも30％長くインキュベートした場合でさえ、まだ結合が＞83％であった。用量を調製した日にちにかかわらず、8分間インキュベートした後でさえ、放射取り込みは、これらの累積したずれによる影響を受けなかった。
【０１０２】
【表８】

V．考察
オペレーターに暴露される放射線を減らすために、全スケール用量の調製の代わりに、より少ないラベル反応を評価した。従って、我々は、本研究において評価された1mCおよび10mCiラベルにより、全スケールの40mCiのプレパレーションを予測されることを立証した。この結果は、1mC〜40mCiの範囲以上において、結合および放射線取り込みに有意差がないことを示した。
【０１０３】
我々は、酢酸ナトリウム、⁹⁰Y塩化物、および結合した抗体の20％量の誤差が、放射ラベルプロトコルにおけるずれの極限値である可能性を示していると判断した。さらに、8分間（通常より3分長い）の培養が有意なプロトコルのずれであると見なされる。一般に、⁹⁰Y塩化物の半減期が短いために、放射性同位体の量は用量調製する日に依存して変更されるであろう。従って、本研究において記載された実験は、用量調製をし得る全範囲を提示するために、⁹⁰Y塩化物を月曜日および金曜日それぞれの両方の濃度において使用して行った。
【０１０４】
20％少ない量の酢酸ナトリウム、かつ8分間のインキュベーションを使用して調製されたY2B8の用量は、標準的ラベル条件と比較して、有意な結合が保持された（＞89％）。同様の結果が、月曜日または金曜日用量プレパレーションから得られた。月曜日または金曜日のいずれにおいても、20％多い量の⁹⁰Y塩化物を添加して、かつ8分までインキュベーションすることにより、標準的ラベル条件と比較して結合が減少された。しかし、結合は、上記の標準化された放射規格の＞90％のままであった。結合は金曜日用量プレパレーションの方がわずかによかった。放射取り込みは、⁹⁰Y塩化物の量の増加によって有意な影響を受けなかった。
【０１０５】
全ての量のずれを同時に起こした場合の影響を評価するために、月曜日または金曜日用量で調製して、2、4、6，および8分のインキュベーション時間を比較した。8分のインキュベーション時間を使用してY2B8が月曜日に調製された場合にのみ、標準化された規格にわずかに結合が合わなかった（標準化された放射規格である86.3％と比較して83.2％）。
【０１０６】
それぞれの引例は、本明細書の参照文献によって援用される。
【図面の簡単な説明】
【図１】A）SB細胞を洗浄して、希釈バッファー（1％ウシ血清アルブミン（BSA）を含む1×PBS、pH7.2-7.4。2B8−MX−DTPA lot#0165A.を使用して調製した2ng/mlのY2B8で3時間インキュベートして、細胞の濃度を増加した）で90×10⁶細胞/mlに懸濁した場合の図。B）細胞濃度対結合した放射活性/総放射活性（B/AT）の二重逆数プロットを示す図。免疫応答性を1/y-intercept×100として推定した。免疫反応性、および相関関数（R）値は、それぞれ77.2％、および0.999であった。[0001]
BACKGROUND OF THE INVENTION
The present invention is a kit and method for radiolabeling proteins and peptides with therapeutic radioisotopes so that these radiolabeled proteins may be administered directly to a patient without the need for further purification. , Kits and methods. Such kits and methods are specifically designed for yttrium-90 (⁹⁰Applicable for labeling proteins and peptides with Y). Furthermore, by optimizing the radiolabel protocol so that there is no need to purify the radiolabeled protein, the present invention enables yttrium labeled drugs to be easily prepared in hospital or outpatient facilities and It fulfilled the long-required demand of those skilled in the art by solving the remaining problem of how to provide a user with an easy-to-understand form to be dosed.
[0002]
[Technical background]
All publications and patent applications in this specification are hereby incorporated by reference herein to the same extent as indicated that each individual publication or patent application was essentially and individually incorporated by reference. Incorporated.
[0003]
Radiolabeled proteins, especially antibodies, have been evaluated for many years as potential diagnostic and therapeutic agents. Since such reagents are believed to be particularly useful as cancer therapeutics, researchers are now beginning to identify tumor-specific antibodies and cognate ligands, or antibodies that bind such antigens. Yes. Directly killing tumor cells by administering a ligand or antibody that specifically binds to a tumor-specific antigen, radiolabeled in combination with a radioisotope with a short range, high energy and abundant particle emission It has the ability to deliver an amount of radiation.
[0004]
In order to target a specific cell type by the particle range of a specific isotope, a label may be selected based on its stability. For example, gamma emitters are commonly used for therapeutic purposes (ie, to visualize tumors and are generally ineffective as lethal drugs). Conversely, alpha and beta emissions will be used to achieve a cell killing effect. Alpha emitters are particularly useful against blood-producing diseases or vascular tumors where they can penetrate well; in some cases, single-particle emitters may be effective enough to kill cells, but generally The alpha emitter must be accurately placed on the cell surface. Conversely, a beta emitter, ie⁹⁰Since Y generally has a longer emission range, they are particularly suitable for larger, more localized diseases.
[0005]
Yttrium-90-labeled antibodies and peptides have shown promising results, especially in clinical treatment protocols (Thomas et al., 1995. Gamma-interferon administration after 90Y radiolabeled antibody therapy: survival and hematopoietic toxicity studies. Int. J Radiat. Oncol. Biol. Phys. 31: 529-534; DeNardo et al. 1995. Yttrium-90 / Indium-111 DOTA peptide chimeric L6: pharmacokinetics, dosimetry and initinal therapeutic studies in patients with breast cancer., J. Nucl Med. 36: 97P). Such conjugates are usually made by linking a protein or antibody to a bifunctional chelator, and then linking the protein construct and the radiolabel via the bifunctional chelator. For example, pending applications 08 / 475,813, 28 / 475,815, and 08 / 478,967, incorporated by reference herein, are radiolabeled to target and extinguish B-cell lymphoma and tumor cells A therapeutic antibody has been described. Conjugated via anti-human CD-20 mouse monoclonal antibody, Y2B, bifunctional chelator MX-DTPA⁹⁰A Y2B8 conjugate comprising Y is specifically disclosed.
[0006]
Patents relating to chelators and chelator conjugates are known to those skilled in the art. For example, Gansow U.S. Patent No. 4,831,175 directs polysubstituted diethylenetriaminepentaacetic acid chelators, and protein conjugates containing the same, and methods for their preparation. Gansow U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 also relate to polysubstituted DTPA chelators. As described by Kozak et al., Several DTPA chelating reagents, including MX-DTPA, have been shown to be suitable for radioimmunotherapy with yttrium-monoclonal antibodies (1989. Nature of the cifunctional chelating agent used for radioimmunotheray). with yttrium-90 monoclonal antibodies: Critical factors in determining in vivo survival and rogan toxicity. cancer Res. 49: 2639-2644). These references are incorporated herein in their entirety.
[0007]
Yttrium-90 is particularly suitable for radioimmunotherapy and radiopeptide therapy for several reasons.⁹⁰Y half-life of 64 hours is a high-energy (E max2.27MeV) pure beta emitter that is long enough to allow the antibody to accumulate in the tumor and has no gamma radiation, for example¹³¹I is different. Its particle emission range is 100-1000 cell diameter. This is a sufficiently small amount of penetrating radiation that can be administered to an outpatient. Furthermore, cell killing does not require internalization of the labeled antibody, and local emission of ionizing radiation is fatal to neighboring tumor cells that may lack the target antigen Should be.
[0008]
However, despite the perceived availability of yttrium-labeled antibodies and the promising clinical consequences of some yttrium-labeled therapies, either radiolabeling or administration to a single site is Because of the inherent difficulties, many patients do not benefit from these treatments. It is clear that this critical problem is that kits and products that can label reagents in situ with alpha and beta emitting radioisotopes are almost completely useless. These would otherwise indicate that such techniques are commercially applicable.
[0009]
The problem in providing a kit for administering a radiolabel and then a therapeutic agent labeled with a destructive isotope is not bone and other targets before such therapeutic agent is administered to the patient There is a long-standing belief in the art that many purification steps are required to remove unbound labels so that free radioactive isotopes that will accumulate in the tissue are not exposed to the patient. It is clear. It is clear that even these kits currently available for labeling antibodies with yttrium require complex purification steps before preparing a therapeutic for administration.
[0010]
For example, Antisoma currently uses the monoclonal antibody HMFG1 (Theragyn®)⁹⁰A kit is provided for post-administration to patients diagnosed with ovarian cancer, labeled Y. Studies extended to Phase I-II showed that this treatment would be particularly beneficial to patients as a follow-up to traditional surgery and chemotherapy (Hird et al., 1993. Ajuvant therapy of ovarian cancer with radioactive monoclonal antibody. Br. J. Cancer 68: 403-406). Furthermore, the Anitisoma labeling method requires removal of unbound labels by Sephadex G50 gel filtration. This is a significant hindrance to the commercial success of Theragyn® label kits, and the treatment is readily available to all uterine cancer patients (patients who benefit from this treatment) It becomes an obstacle to make it possible.
[0011]
The fact that such reagents currently require column purification prior to administration is this, unless a simplified method is provided that allows a physician to administer quickly, efficiently and safely. The availability to all patients who would benefit from modern technology was and was primarily an obstacle. For example, an outpatient physician does not have the time or facilities to purify a drug by HPLC or gel filtration chromatography before administering the drug to the patient. This means that additional equipment must be available as a place for simultaneous drug production and immediate delivery to the physician. This dramatically increases the cost of treatment and in some cases may require the patient to travel quite far to receive treatment. Alternatively, the drug could be labeled outside the facility. This will require the therapeutic agent to be prepared in advance and stored for at least a short period of time. This not only affects the decrease in radioisotope intensity through radioactive decay upon storage, but also causes significant damage to the structural integrity of the protein by being overexposed to the radioisotope.
[0012]
For example,⁹⁰The radiolytic properties of Y and similar radioisotopes have been discussed in many reports (ie, Salako et al., 1998. Effects of radiolysis on yttrium-90-labeled Lym-1 antibody preparations. J. Nucl. Med). .39: 667-670; Chakrabarti et al. 1996. Prevention of radiolysis of monoclonal antibody during labeling .J. Nucl. Med. 37: 1384-1388). As Chakrabarti et al. Stated,⁹⁰Radionuclides such as Y deliver large amounts of radiation to the antibody during the labeling process as well as during storage. Talkingly, selective targeting of tumor cells was ignored, and it was possible to expose tissues that were not targeted to significant levels of toxicity. The cause of antibody damage was caused by radiation.
[0013]
The mechanism of radiation damage is due to the generation of free radicals (Pizzarello. 1975. Direct and indirect action. In: Pizzarello and Witcofski, eds. Basic Radiation Biology, 2nd ed. Philadeophia: Lea and Febger, pp. 20- 29). However, as Salako et al. Stated, at 2.2 MeV energy,⁹⁰Beta particles emitted from Y will be able to easily break most chemical bonds, including antibody disulfide bonds (Skoog. 1985. Principles of Instrumental Analysis, 3rd edition. San Francisco: Saunders). Therefore, the labeled protein is⁹⁰The shorter the exposure time to a destructive radioisotope such as Y, the more structural integrity and the specificity of binding required for the protein to interact with the target antigen, when it reaches the target site after administration There is a high probability of keeping up.
[0014]
⁹⁰The radiolytic properties of Y have been known to those skilled in the art for many years, and when these treatments are used commercially,⁹⁰Many attempts have been made to solve the problems that exist in Y. For example, both Salako et al. And Chakrabarti et al.⁹⁰The use of radiation protection material in the preparation of Y-labeled antibodies was evaluated. In particular, Salako et al.⁹⁰It has been reported that the immunoactivity of Y-labeled antibodies can be maintained up to 72 hours. However, the specific activity demonstrated by Sadako preparation was rather low (less than 2 mCi / ml). Furthermore, Salako or Chakrabarti do not report any effort prior to the numerous purification steps required after labeling the antibody. Salako et al. Label for 45 minutes to 1 hour and then purify the antibody by molecular sieve chromatography, while Chakrabarti labels for nearly 3 hours and purifies by gel filtration chromatography. Both of these methods⁹⁰It will not be a means to bring Y-labeled therapeutics to outpatient facilities.
[0015]
Chinol and Hnatowich made with their own generator⁹⁰Use Y to have specific activity in the range of 1-3 mCi / mg⁹⁰For proteins labeled with Y, 90% radiochemical purity could be obtained without purification after labeling (1987. Generator ^ produced yttrium-90 for radioimmunotherapy. J. Nucl. Med. 28 (9): 1465 -1470). However, the authors were particularly opposed to administering preparations with a purity of less than 95% to patients, and showed that HPLC would be an important and "probably essential" step.
[0016]
Those who recognize that HPLC and other types of purification methods must have been removed from outpatient and hospital equipment maintain high levels of label incorporation and maintain an acceptable level of antibody stability. like,⁹⁰Development of a protocol that fully labels Y was not successful. If high levels of radiouptake are not consistently achieved, the patient will be exposed to unacceptable levels of unbound free radiolabel if this label is not purified from the reagent. Once again, if the integrity of the antibody structure is damaged such that the antibody loses specificity for the target, such a reagent will not be able to specifically bind its cognate ligand.
[0017]
Mather and colleagues have used tumor-specific antibodies in a way that avoids purification after labeling.⁹⁰Started for the purpose of labeling with Y (1989. Labeling monoclonal antibodies with yttrium-90. Eur. J. Nucl. Med. 15: 307-312). However, Mather found that high-efficiency labels (greater than 95%) can be obtained only with loose specific activity (1 mCi / mg). Furthermore, Mather et al. Report that their antibody preparations showed signs of destruction (by radiation) after several hours. This may be because Mather et al. Performed their labeling reactions for over an hour, as many others in the field do.
[0018]
For example, without further purification steps,¹¹¹There are proposed methods for labeling proteins with less destructive reagents such as In. Richardson et al. Have developed such a kit to facilitate the construction of a kit for use in diagnosis.¹¹¹Propose a method for labeling antibodies with In (Richardson et al. 1987. Optimization and batvh production of DTPA-labeled antibody kits for routine use in¹¹¹In immunoscintography. Nucl. Med. Comm. 8: 347-356). However, the labeling method proposed by Richardson et al. Is performed for over an hour and does not undergo radiolysis.¹¹¹While it may be possible in In, as demonstrated by the difficulties reported by Mather et al.⁹⁰Y does not seem to be accepted.
[0019]
This brings us surprises and unexpected benefits of the present invention. This is the protein⁹⁰In the method of radiolabeling with Y, it provides valuable insight not recognized by others skilled in the art. Surprisingly, we increase the specific activity of HPLC, or other purification steps, and those reagents that others have long believed to be essential for obtaining pure reagents. The long incubation period adopted by others for⁹⁰It has been found to be detrimental to the method of preparing Y-labeled reagents. Such time-inclusive methods only increase protein damage due to radiolysis, increasing the rate of proteolysis by the time it is ready to inject low specificity and radiolabeled protein Is done. Surprisingly, the present invention⁹⁰Efficient labeling with Y can be done in as little as two to five minutes, and in fact such a label loses its efficiency even if the reaction time only increases by more than eight minutes.
[0020]
Now, by the method of the present invention,⁹⁰The fact that labeling with Y may be accomplished in as little as two minutes will completely resolve current skepticism in the field of applicability of kits that radiolabel yttrium in hospital and outpatient facilities. Thus, the kits of the present invention are likely to have long and urgent requirements recognized by many cancer patients and physicians, as well as commercial applicability and the availability of protein-based radiolabeled cancer therapeutics. Will eventually meet the long and urgent demands on.
[0021]
SUMMARY OF THE INVENTION
The present invention relates to methods and kits for radiolabeling chelator-binding proteins or peptides with therapeutic radioisotopes for administration to a patient. The method of the present invention essentially consists of
(I) mixing a chelator-binding protein or peptide with a solution containing a radioisotope or salt thereof; and
(Ii) a radiolabeled antibody having sufficient purity, ie, level of radiouptake, specific activity, and binding specificity, such that the radiolabeled antibody may be administered directly to a patient without further production. Incubating the mixture for a sufficient amount of time under good conditions to obtain a suitable protein or peptide;
It is a method including.
[0022]
The kit of the present invention essentially consists of
(I) a vial containing a chelator-binding protein or peptide in a buffer;
(Ii) a vial containing a formulation buffer for stabilizing the radiolabeled antibody and administering to the patient; and
(Iii) instructions for carrying out the radiolabeling method when the radiolabeled protein or peptide is exposed to a radioisotope or salt thereof under good conditions as recommended in the instructions Sufficient purity, specific activity, and binding specificity such that the radiolabeled antibody may be diluted to an appropriate concentration in formulation buffer and administered directly to the patient without further purification Instructions to obtain a radiolabeled protein or peptide having
It is a kit containing.
[0023]
Detailed Description of the Invention
Unless defined otherwise, all technical and chemical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and articles similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and articles are described.
[0024]
A method for radiolabeling a chelator binding protein or peptide with a therapeutic radioisotope for administration to a patient comprising:
(I) mixing a chelator-binding protein or peptide with a solution containing a radioisotope or salt thereof;
(Ii) a radiolabeled antibody having sufficient purity, ie, level of radiouptake, specific activity, and binding specificity, such that the radiolabeled antibody may be administered directly to a patient without further production. Incubating the mixture for a sufficient amount of time under good conditions to obtain a suitable protein or peptide,
The present invention encompasses methods comprising: “Further purification” includes HPLC, gel filtration, other types of column chromatography, and other separation techniques used to remove free or bound unbound radiolabels.
[0025]
The methods of the present invention are particularly applicable to therapeutic radioisotopes that may be dangerous for the structural integrity of the protein, generally by being radiolytic. Such therapeutic radioisotopes are generally selected from the group consisting of alpha and beta emitters. Preferred therapeutic radionuclides include²⁰³Pb,²¹²Pb,²¹²Bi,¹⁰⁹Pd,⁶⁴Cu,⁹⁰Y,⁷⁷Br,^{211 ,}A,⁹⁷Ru,¹⁰⁵Rh,¹⁹⁵Au and¹⁷⁷Includes Lu. Other radionuclides with therapeutic utility are described in U.S. Patent 5,541,287, incorporated herein by reference. Particularly preferred radionuclides are⁹⁰Y,⁶⁴Cu,¹³¹I,¹⁸⁶Re, and¹⁸⁸It is a powerful beta emitter such as Re. These will cause intramolecular decay. “Therapeutic” radioactive isotopes generally refer to radioactive isotopes such as beta and gamma emitters which have cytotoxic effects, to the extent that such radioactive isotopes will also be used for diagnostic purposes. For these purposes, these isotopes are not excluded from the scope of the present invention because the radiolytic properties of these isotopes are optimal for the disclosed methods and kits.
[0026]
The methods of the present invention may be used to label proteins or peptides, particularly those that must maintain structural integrity, due to target specificity. Preferred proteins are antibodies that recognize tumor-specific or tumor-associated antigens, or antibody fragments such as Fab, (Fab) 2 and Fv fragments. Preferred peptides include somatostatin, vasoactive intestinal peptide (VIP), substance P, and other peptides that bind to cell receptors. Such peptides, and chelator-binding derivatives of such peptides are disclosed in U.S. Patent No. 5,830,431, incorporated herein by reference.
[0027]
The “sufficient incubation time” mentioned in the method of the invention is that when the reagent is of sufficient radio uptake and radiochemical purity that it may be administered directly to the patient without the need for further purification. This is an acceptable time range. Sufficient radio uptake and purity are generally recognized by those skilled in the art to be at least 95%, but may vary depending on the toxicity of the label. It should also be apparent to those skilled in the art that the range of radiation uptake considered sufficient is a function at the required level of efficiency.⁹⁰Y, and especially of the present invention⁹⁰For antibodies labeled with Y, such sufficient time is generally less than about 8 minutes, and more preferably about 2 to about 5 minutes, and the chelator and protein are sensitive when the chelator-binding protein is labeled. The molar ratio to react with is given.
[0028]
It should be apparent to those skilled in the art that the optimal time required to label a particular protein may vary depending on the protein, the particular radiolabel, and the particular complex used. The underlying factor in optimizing the time spent for radiolabeling is the ratio of chelator to protein in the labeled reagent. For example, to achieve a therapeutically useful level of uptake, the ratio of chelator to protein must be high enough (ie 95%), but high enough to compromise the structural integrity of the protein or the immunoreactivity. Don't pass too much. This requires a balanced process that in some cases is a low level binding chelator and labels for a long time.
[0029]
For example, we have achieved a desired level of purity.⁹⁰To label with Y, MX-DTPA is used as the chelator for 5 minutes and the molar ratio of chelator to antibody is about 1¹/₂I found that it would be done only at ~ 1. Although the ratio of chelator to antibody can actually be increased, this was not necessary because the desired level of radiouptake and specific activity was obtained after a brief labeling. Given this discovery, parameters such as chelator and protein concentrations can be determined by the empirical methods of one skilled in the art for other proteins and peptides, the sites available for therapeutic label selection, chelator selection, and chelator binding. Depending on the number of proteins, the sensitivity of the protein to radiolysis, the level of efficiency desired, etc.
[0030]
Any bifunctional chelator may be used in the methods of the present invention so long as it can bind to both the protein of interest and the radioisotope. Preferred chelators are selected from the group consisting of MX-DTPA, phenyl-DTPA, benzyl-DTPA, CHX-DTPA, DOTA and derivatives thereof. A particularly preferred chelator is MX-DTPA.
[0031]
As stated in the method of the present invention, “good conditions” include acceptable temperature, pH, and buffer conditions. It should be apparent to those skilled in the art that reaction conditions that are inhibitory or detrimental to the labeling reaction should not be selected. Lewis et al. Discuss the reaction conditions to be considered when radiolabeling immune complexes. This is incorporated herein by reference (1994.A facile, water-soluble method for modification of protein with DOTA.Use of elevated temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled immunoconjugate. Bioconjugate Chem. 5: 565-576).
[0032]
In the reaction, the acceptable temperature may vary depending on the labeled protein, but the general range is from about 25 ° C to about 43 ° C. Lewis et al. Found that increasing the temperature of the radiolabel reaction from 25 ° C to 43 ° C increased both the efficiency of radiometal incorporation and the rate of stabilization of the DOTA radiocomplex investigated.
[0033]
The acceptable pH may vary considerably depending on the radiolabel used. The different recommended pH values for labels are generally known to those skilled in the art and may be selected accordingly by the radioisotope. For example,⁹⁰An acceptable pH for Y is about 3 to 6, but more preferably about 4.2.
[0034]
Acceptable buffers will also vary depending on the particular label. For example, Lewis et al. And others believe that citric acid is present⁹⁰It was discovered that the labeling reaction by Y is inhibited. Therefore, the radiation label⁹⁰If Y is selected, citrate buffer may not be suitable.⁹⁰When labeled with Y, preferred buffers are acetate buffer, and more preferably sodium acetate buffer at a concentration between about 10 and about 1000 mM.
[0035]
A mild (non-hazardous) radioprotectant may be included in the reaction buffer provided it does not inhibit or otherwise adversely affect the labeling reaction. According to Chakrabarti, ascorbic acid is one such radioprotectant that does not affect the labeling reaction. However, when human serum albumin is used for the labeling reaction, it should be noted that there are metals that affect the labeling process.
[0036]
Since the present invention is particularly concerned with radiolabeling proteins with radiolytic isopologues, there is a certain balance between binding specificity and specific activity encountered by those skilled in the art when practicing the present invention. might exist. For example, if the specific activity is very high (ie, 5 mCi / mg or more, preferably 10 mCi / mg or more, and more preferably 15 mCi / mg or more), a protein construct with the desired binding specificity is sufficient in the tumor area. Will have the ability to kill. However, the population of proteins that retain their immunoreactivity as a whole may be lower than the population with low specific activity due to radiolysis of the radiolabel. One skilled in the art may make a selection that impairs a certain level of immunoreactivity, depending on the level of specific activity desired.
[0037]
For example, the inventor⁹⁰It has been discovered that when an antibody is labeled with a specific activity of about 15 mCi / mg with Y, the binding specificity, or protein immunoreactivity, is generally at least about 70%. In this case, it may vary depending on the sensitivity of the antibody and the radiolytic nature of the radioisotope used, and if a high level of immunoreactivity or specific activity is desired, a skilled expert May be operated by. The inventors have⁹⁰Specific activity up to about 20 mCi / mg was obtained with Y. For therapeutic application, at least 50% binding activity is desirable.
[0038]
If desired, a binding assay that may be used to assess the percent binding affinity after labeling, and the immunoreactivity of the complex, was recognized simultaneously with this and was filed in And 09 /. After the labeling method of the present invention, no further purification is required, but a TLC-based assay to confirm the level of radiation uptake should always be performed so as not to jeopardize patient health. . Such an assay can be performed in about 3-4 minutes and should not significantly affect the stability or efficiency of the radiation therapy.
[0039]
The present invention also provides a kit for radiolabeling a chelator-binding protein or peptide with a therapeutic radioisotope for administration to a patient,
The kit of the present invention essentially consists of
(I) a vial containing a chelator-binding protein or peptide in a buffer;
(Ii) a vial containing a formulation buffer for stabilizing the radiolabeled antibody and administering to the patient; and
(Iii) instructions for carrying out the radiolabeling method when the radiolabeled protein or peptide is exposed to a radioisotope or salt thereof under good conditions as recommended in the instructions Sufficient purity, specific activity, and binding specificity such that the radiolabeled antibody may be diluted to an appropriate concentration in formulation buffer and administered directly to the patient without further purification Instructions to obtain a radiolabeled protein or peptide having
Is included. The chelator binding protein or peptide may be supplied in lyophilized form.
[0040]
It should be understood that the kit of the present invention was designed to perform the methods described herein and may therefore be used for that purpose. Therefore, when reading the present invention, it is to be understood that the kit instructions will be based on the above method, and that, when considered with respect to this kit, the above addressed issues have the same relevance and meaning. , Should be obvious to those involved. Moreover, when reading this disclosure, it should be apparent that other kit embodiments as a whole are encompassed by the present invention. These will include compositions such as acetate buffer to adjust the pH of the radiolabel as described above.
[0041]
A particularly advantageous composition of the kit is a formulation buffer for stabilizing against the effects of radiolysis and for administering a radiolabeled antibody to a patient. The formulation buffer is a pharmaceutically acceptable carrier that serves both to dilute the labeled antibody and as a dosing buffer. Although pharmaceutically acceptable diluents may be used to administer therapeutic or diagnostic antibodies, the formulation buffers of the present invention are particularly suitable for radiolabeled antibodies.
[0042]
For example, the formulation buffer of the present invention includes a radioprotectant that minimizes radiolysis by yttrium and other powerful radionuclides, such as human serum albumin (HAS) or ascorbic acid. Other radioprotectors are known to those skilled in the art and could be used as the formulation buffer of the present invention. Free radical scavengers (phenol, sulfite, glutathione, cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP (: O) H2, glycerol, sodium formaldehydesulfoxylate, Na₂S₂O_Five, Na₂S₂O_Three, And SO₂Etc.).
[0043]
The formulation buffer of the present invention also contains an excess amount of unbound chelator. The purpose of including an unbound chelator is that the chelator is a “osteogenic” isotope, i.e., as a scavenger to any non-protein-bound radiolabel in the patient and by expelling the radiolabel, i.e.⁹⁰It is useful to reduce bone uptake in Y patients. For example, if the kit antibody is conjugated to a DTPA chelator, either excess DTPA or other chelator may be included in the formulation buffer. The formulation buffer is also preferably provided in an amount such that the entire dose is transferred to the reaction vial. As discussed above, this results in increased ease of use and reproducibility because the exact amount does not have to be measured and transferred.
[0044]
Preferred formulation buffers include phosphate buffer or saline, human serum albumin, and DTPA. The human serum albumin is preferably at a concentration between about 5-25% (w / v), and more preferably at a concentration of about 7.5% (w / v). The concentration of the DTPA is preferably about 1 mM. Ascorbate may be used as a substitute for human serum albumin and is generally used at a concentration of about 1-100 mg / ml. However, a wider range of concentrations may be used so as not to compromise patient safety.
[0045]
The kit may be supplied as another alternative depending on the preference of the purchaser. For example, the kit further includes a sterile reaction vial in which both the labeling reaction and the dilution of the formulation buffer will be performed. In order to reduce waste, further embodiments are envisioned, where a buffer for adjusting the pH of the radiolabel is provided in the actual reaction vial. It may be possible to order a label kit in advance and then order a radioisotope immediately prior to administration, although kits that further include a radioactive isotope vial are also contemplated. A kit containing a second protein or peptide vial is also conceivable, used as a control in assessing the binding affinity of the radiolabeled product or, in some cases, a combination of radiolabeled protein or peptide and therapy To contribute as one to.
[0046]
[Detailed Description of Preferred Embodiments]
In clinical trials to treat recurrent B-cell lymphoma,⁹⁰A Y-labeled anti-CD20 mouse monoclonal antibody (Y2B8) is currently being evaluated. The 2B8 antibody is a mouse antibody that recognizes human CD20. Recently, a chimeric version of this antibody (RITUXAN®) was approved by the FDA for the treatment of non-Hodgkin lymphoma. U.S. Patent Application No. 08 / 475,813, which is hereby incorporated by reference, has the disclosure of administering RITUXAN (R) over time with a mouse monoclonal antibody labeled with yttrium in combination with therapeutic therapy. Administration of yttrium-labeled anti-CD20 antibody after administration of RITUXAN (R) (A) to remove any peripheral blood B cells remaining without being removed by the anti-CD20 chimeric antibody; (b) It was sufficient to initiate removal of B cells from the lymph nodes; or (c) to initiate removal of B cells from other tissues.
[0047]
Therefore, the effectiveness of anti-CD20 antibodies for the treatment of non-Hodgkin's lymphoma has been improved and the sensitivity of lymphocytes to radioactivity has become known so that such therapeutic antibodies can be made commercially available in kit form. Will be very useful. This facilitates modification with radiolabels in clinical facilities and will be administered directly to the patient.
[0048]
A kit that radiolabels the 2B8 antibody preferably comprises four components:
1.) 2mg / ml 2B8-MX-DTPA low metal normal saline,
2.) 50 mM sodium acetate used to adjust the radioisotope solution to the optimum pH for the label,
3.) Formulation buffer (7.5% (w / v) human serum albumin and 1 mM PBS containing 1 mM PBS, pH 7.4), and optionally
4.) Empty 10 mL glass vial (reaction vial) (the “10 mL” reaction vial is actually held without inconvenience and technically slightly larger than “10 mL”). All components have been tested to be sterilized and pyrogen free.
[0049]
In this section, validation of this radioisotope labeling kit (to produce antibodies that are simple and easy to use and that are radiolabeled with ≧ 95% radiouptake and that retains binding to antigen-positive cells acceptably) To summarize. Experimental parameters that affect binding and radiation uptake were also evaluated.
[0050]
【Example】
Example 1. 2B8⁹⁰Radiation label kit and method for labeling with Y
A. Reagent for radiolabel kit.
[0051]
1. 2B8-MX-DTPA, IDEC; Lot # 082395RM2,
2. 50 mM sodium acetate, low metal, IDEL; Lot # 082395RM3,
3. Formulation buffer (7.5% (w / v) human serum albumin and 1 mM DTPA, 1 × PBS, pH 7.4), IDEC. Lot # 082395RM1,
4. Reaction vial, 10mL, IDEC,
B. Materials and equipment.
[0052]
1. Biotex Tec-Control Radioincorporation Kit, Cat. # 151-770,
2. Gloves: powder free,
3. Sterile polypropylene syringe,
4. Sterile needle,
5. Small tube with lid; 1.5ml,
C. Method.
[0053]
1. Preparation of Y2B8 using radiolabel kit
Kit reagents were prepared and filled into septal glass vials. Type I borosilicate vials (2 or 10 ml) were rinsed with sterile water for injection (WFI) and autoclaved before use. Reagents were manually filled, crimped in a Class 100 room, and tested for pyrogenicity and sterility using the USP method.
[0054]
Auxiliary reagents:
1. Yttrium- [90]: Chloride salt, carrier free, HCl solution.
[0055]
Precautionary measures
1. All steps should be performed using aseptic technique.
2. Radiolabel kit components should be at room temperature prior to use.
[0056]
Radiation label protocol
1. Add to reaction vial⁹⁰YCL_ThreeThe amount of was estimated as follows
a. Radioactive concentration at radiolabel:
C₀= Radioactivity concentration at the time of calibration (see analysis method of manufacturer's certificate),
Δt = change time (positive number after calibration, negative number before calibration),
Radioactive concentration at the time of labeling = C₀/e0.0108 (Δt).
[0057]
b. Add to reaction vial⁹⁰YCL_ThreeAmount of
Radioactive concentration at 45 mCi / label = amount added to reaction vial.
[0058]
2. The amount of 50 mM sodium acetate added to the reaction vial was estimated as follows:
a.0.040M in HCl (Amersham)⁹⁰YCL_ThreeTo:
⁹⁰YCL_ThreeAmount of (step 1b) × (0.8) = amount of sodium acetate to be added,
b.0.050M in HCl (Nordion)⁹⁰YCL_ThreeTo:
⁹⁰YCL_ThreeAmount of (step 1b) × (1.0) = amount of sodium acetate to be added,
3. Septal reaction vials and sodium acetate vials were wiped with alcohol. Using a 1 cc syringe, an estimated amount (Step 1a or 1b) of 50 mM sodium acetate (Step 2) was transferred to the reaction vial. The vial was inverted and mixed several times.
[0059]
Four. ⁹⁰YCL_ThreeThe septum of the source vial was wiped with alcohol. The needle and vial were mounted on a sterilized 0.2 μm filter. Invert the 1cc syringe and use the required amount (step 1b)⁹⁰YCL_ThreeWas transferred to a reaction vial. The vial was inverted and mixed several times.
[0060]
5. The septum of the 2B8-MX-DTPA vial was wiped with alcohol. Using a 3 cc syringe, 1.5 ml of 2B8-MX-DTPA was transferred to the reaction vial. The vial was inverted and mixed several times.
[0061]
6. The total amount of the reaction mixture was the amount of Y-90 chloride added (step 4), the amount of 50 mM sodium acetate added (step 3), and the amount of 2B8-MX-DTPA added (step 5). ) And estimated.
[0062]
7. To make the final volume of 10 mL, the amount of formulation buffer added to the reaction vial was estimated by subtracting the total reaction volume estimated in step 6 from 10.
[0063]
8. The formulation buffer vial was wiped with alcohol and the vial was vented. The reaction vial was attached to a 0.2 μm syringe filter using a needle based on the viscosity of the formulation buffer. Using a 10 cc sterile syringe equipped with an appropriate gauge needle, the amount of formulation buffer estimated in step 7 was transferred to the reaction vial. The vent needle was removed from the reaction vial and the vial was inverted several times to mix (final product). The vials were incubated for at least 5 minutes before performing the “radioactivity assay”. The solution color is amber, confirming that the formulation buffer has been added by filling the reaction vial.
[0064]
9. Total radioactivity in the final product vial,⁹⁰Measurements were made using an appropriate instrument set for measuring Y.
[0065]
10. The final product was immediately brought to 2 ° C to 8 ° C and stored until needed for patient administration.
[0066]
2. Radiation uptake assay
% Radioincorporation was measured by instantaneous thin layer chromatography (ITLC) using the Biodex Tec-Control Radiochromatographic Kit according to the following protocol.
[0067]
Auxiliary materials and equipment:
1.⁹⁰2B8-MX-DTPA radiolabeled with Y,
2. Tube for measuring radioactive TLC strips,
3. Scissors,
4. Sterile syringe; 1cc,
5. Sterilization needle; 26G,
6. Gamma counter or scintillation counter,
7. Pipettor. ,
procedure
1. The entire Biodex operating manual should be read first.
2. Each radiolabeled sample was tested in triplicate according to kit instructions; one strip was developed per vial.
3. To spot the radiolabeled sample on the chromatography strip, 1 μl was spotted on the starting line with a pipettor. Alternatively, a small drop taken from a 26G needle attached to a 1 cc sterile syringe may be spotted. The antibody remains at the starting point and is not taken up⁹⁰Y-DTPA moves to the tip of the solvent.
4. Appropriate counter, ie⁹⁰The background was corrected using a scintillation counter for Y, and the activity of each fraction was measured.
5. The ratio of radiolabeled antibody was estimated according to the Biodex instrument.
3. Binding assay
Auxiliary reagent
1.⁹⁰Y2B8-MX-DTPA
2. Lyophilized cells
Human cell lines sb (CD20 positive) and HSP (CD20 negative) were obtained from the American Type Culture Collection using RPMI-1640 containing 10% fetal bovine serum supplemented with 2% glutamine. Incubated in flask. Culture medium is 37 ° C and 5% CO₂Maintained at. Cells are generally divided into 1: 2, 0.5-2.5 × 10⁶Cells / ml and viability were collected> 80%. Cell concentration was measured using a hemocytometer and viability was measured by trypan blue exclusion.
0.5-2 × 10⁶Cells at a cell concentration of cells / ml were harvested by centrifugation at ambient temperature and washed twice with 1 × HBSS. Pellet cells in 1 x HBSS containing 1% (w / v) bovine serum albumin (BSA) and 10% (w / v) mannitol (lyophilized buffer), l50 x 10⁶Resuspended as cells / ml, dispense 0.5 ml into a 1.5 ml polypropylene microfuge tube of O-ring gasket, stored at -70 degrees and lyophilized overnight at 30-60 mTorr. Lyophilized cell tubes were dried and stored at 2-8 ° C. and reconstituted with sterile water for the assay; lyophilized cell tubes in microfuge tubes were stored dry.
[0068]
3. Sterile water for cleaning or sterile water for injection.
4. Dilution buffer (1 × PBS with 1% bovine serum albumin (BSA) and 0.02% sodium azide, pH 7.2-7.4).
[0069]
Method
Preparation of radiolabeled antibody samples
1. Obtained radiolabeled antibodies stored at 2-8 ° C.
2. A 10 μl volume was removed with P20 and added to a 1.5 ml macrofuge tube containing 990 μl of dilution buffer (1: 100 dilution). The tip was rinsed and the tube was vortexed lightly.
3. A 50 ml sterile polypropylene tube with lid was obtained and 10 ml of dilution buffer was placed in the tube using a 10 ml serum pipette.
4. Remove 35 μl volume from 1: 100 dilution tube with p200 and add to conical tube containing 10 ml dilution buffer. Mix well.
Preparation of lyophilized cells
1. Three lyophilized SB cell tubes were prepared.
2. A 0.5 ml volume of SWFI was added to each tube and the tubes vortexed until a single cell suspension was obtained.
3. Three empty microfuge tubes were prepared; 0.5 ml of dilution buffer was added to the three tubes to present a cell free control.
[0070]
Assay protocol
1. 0.5ml diluted⁹⁰Y2B8-MX-DTPA was added to each tube.
2. After confirming that the lid was securely closed, the tube was left on the mixer for 45 minutes.
3. After 45 minutes of incubation at ambient temperature, the cells were centrifuged for 5 minutes to pellet.
4. 0.8 ml of the supernatant was transferred to a scintillation vial.
5. Scintillation cocktail was added to each vial.
6. The amount of radioactivity in the vial was measured using a scintillation counter with modified background.
[0071]
D results
The reproducibility and difficulty of the Y2B8 radiolabel protocol was evaluated by performing several validation experiments using different lots for each radioisotope. Six confirmation lots were prepared with each Y2B8 by five operators. These lots were named as follows and performed on the following equipment:
# 1: IDEC medicine,
# 2: IDEC medicine,
# 3: IDEC medicine,
# 4: MD Anderson Health Center,
# 5: Mayo Clnic,
# 6: City of Hope,
Table 1 summarizes the results of testing against these biconfirmation lots.
[0072]
[Table 1]

For the six confirmed lots prepared, the percentage of binding obtained is in the range of 78.6% to 87.0% with an average of 82.3%. The average radiation uptake value for Y2B8 is 98.8% (range 96.3% to 99.5%). Overall, these results confirm the reproducibility and difficulty of preparing Y2B8 using the radioisotope label kit method, and overall, Y2B8 prepared using this radiolabel kit is It is suitable for use.
[0073]
Example 2. Initial evaluation of reaction parameters-pH and reaction time
After performing the labeling reaction under various pH and reaction time conditions, an initial rate experiment was conducted.⁹⁰Radiouptake and binding of Y-labeled antibodies was evaluated. When radiolabeled by incubation for 5 minutes in the pH range from 3.9 to 4.7,> 80% had> 96% radiouptake while maintaining binding to CD20 positive cells (Table 2). . Similar results were obtained when incubated for 3, 5, and 10 minutes at pH 2.9 to 4.6 (Table 3).
[0074]
[Table 2]

[0075]
[Table 3]

The immunoreactivity of Y2B8 preparation was measured using the method of Lindmo et al. The amount of freshly recovered CD20 positive SB cells was increased and incubated with a fixed amount of Y2B8 under antigen-excess conditions. Analysis of binding data by reciprocal plots showed that Y2B8 immunoreactivity was 72.2% after a single preparation (Figure 1).
[0076]
Example 3. Evaluation of further reaction parameters
I Introduction
The experiments described in this section investigated the impact on protocol drift for Y2B8 binding prepared using the Y2B8 radiolabel kit. The binding of radiolabeled antibodies will be affected by several parameters in the process of radiolabeling (Table 4).
[0077]
[Table 4]

The following deviations from the radiolabel protocol were identified and included as having the most negative impact on binding:
1.) Addition of less amount of sodium acetate,
2.) Addition of excess chloride solution,
3.) Addition of smaller amount of 2B8-MX-DTPA, and
4.) Incubation exceeding the maximum reaction time,
It is. The effects of these deviations were evaluated separately or simultaneously.
[0078]
When assessed separately, even when incubated for 8 minutes, the 20% amount of deviation in 1-3 of the above items resulted in IDEC-Y2B8 passing the release specification established for binding in clinical trials. became.
[0079]
In studies that caused all three deviations simultaneously, for doses prepared using the Monday label protocol (maximum possible radiolysis) and incubated for 8 minutes (60% longer than normal) Only slightly below clinical release specifications (<3%). Conversely, doses adjusted using the Friday label protocol maintained acceptable binding results in spite of the cumulative effects of deviations in all four parameters (1-4 above). Radiation uptake was above 95% of the clinical release specification when it occurred separately and simultaneously in all deviations.
[0080]
II. Parameter selection
We accept a maximum deviation from the protocol used in radiopharmaceuticals that allows for a 20% deviation in the amount of reagent required or a 30% excess of the reaction time of up to 6 minutes normally used. Judged to show. In this study, we evaluated the effects of these shifts on IDEC-Y2B8 binding. We simulated the “Monday” and “Friday” labels to demonstrate that the conditions evaluated exhibited extremes of dose adjustment throughout the week. The effect of the combination on binding when all deviations occur in a single dose preparation; and⁹⁰The effect of these deviations on Y radiation uptake was evaluated.
[0081]
The “Monday” and “Friday” labels reflect the following:⁹⁰Since the Y chloride solution has a short half-life (64 h), the amount of radioisotope used depends on the day of the week when the dose was prepared. For this reason, the amount of response prepared on Monday is less, and as a result⁹⁰The concentration of Y will be higher, possibly resulting in stronger radiolysis. Therefore, we have simulated the Monday and Friday labeling method and have demonstrated the extremes of dose preparation throughout the week.
[0082]
III. Materials and Methods
A. Reagent
1.0.05M in HCl⁹⁰YCl; Pacific Northwest National Laboratory, reagent grade; P.O. # 08016,08118,
2. Ultrex HAL; J.T. Baker, Product # 6900, Lot # J22539,
3. Sterile water for cleaning; Baxter, Part # 2F7114, Lot # G926092,
4. Dilution buffer: pH 7.4 with 10 mM PBS, 1% BSA; Sigma, Part # P-3688, Lot # 076H8913,
5. IDEC Supplied Radiolabeling kit; IDEC Part # 130018, Lot # 0129, including:
a.) 2B8-MX-DTPA; IDECPart # 129017, Lot # 0165,
b.) 50 mM sodium acetate; IDEL Part # 121017, Lot # 0209A,
c.) Formulation buffer; IDEC Part # 120015, Lot # 0202,
d.) Reaction vial; IDEC Part # 122015, Lot # 0218,
6. Lyophilized SB cells, IDEC Part # 127, Lot # 127-001F,
B. Materials and equipment.
[0083]
1. Pipetter (20, 200, and 1000gL),
2. Vortex,
3. Metal-free pipette tips (Biorad; metal-free),
4. Gamma counter (Isodata, Model # 20-10),
5. Glass tube (12 x 75mm),
6. Polypropylene tube (Costar; 15mL and 50mL conical, sterile),
7. Tec-Control Radiochromatographic Kit (BioDex; Cat # 151-770),
8. Micro centrifuge (Savant),
9. Polypropylene microfuge tube, metal free (Biorad; Cat # 223-9780).
[0084]
C. Method
1. Preparation of Y2B8
Typically, ⁹⁰Y-labeled 2B8-MX-DTPA was prepared using the small scale version of the radiolabel kit protocol described above, modified with the changes described below. Radiolabels have a storage concentration of 84 mCi / mL or 29.8 mCi / mL.⁹⁰Y chloride was used to simulate a Monday or Friday dose preparation, respectively (based on a Wednesday calibration of 50 mCi / mL). Concentrated⁹⁰The Y chloride solution was diluted in a “metal free” plastic microfuge tube using 50 mM HCl (Ultrex, high-purity). Ultrex (high-purity) HCl was diluted to 50 mM with sterile water for washing (SWFI). Radiolabel reactions were performed in “metal free” plastic microfuge tubes, 15 ml conical tubes, or 10 mL glass septal reaction vials provided by the Y2B8 radiolabel kit.
[0085]
a. Adjust full scale dose from small scale label
Radiolabel reactions of 0, 3, 10, and 40 mCi were performed using reaction conditions that simulated Monday dose preparation. The reagent amount of each reaction was summarized in Table 5 as mLs.
[0086]
[Table 5]

After 5 minutes incubation, 20 μl sample was removed and the final antibody concentration was diluted to 0.21 mg / mL with formulation buffer and stored at 2-8 ° C. until assayed. In all the following experiments described in this report, 1 mCi reaction was used as a control, so the binding value was normalized to 1 mCi reaction. Reported values were normalized as 1 mCi control samples by dividing the binding value for each reaction by the binding value for the control and displayed as a percentage.
[0087]
b. Effect on the amount of sodium acetate added
On the Monday label, 10mCi⁹⁰Y chloride (0.119 mL) and 0.114 mL of 50 mM sodium acetate were mixed. This amount of 50 mM sodium acetate is 20% less than the amount of buffer normally used to adjust doses in the IDEC-Y2B8 clinic. Bound antibody (2B8-MX-DTPA) was added (0.333 ml), the sample was mixed and then incubated at ambient temperature. The specific activity of the radiolabel solution was 18.9 mCi / mg antibody. At 2 minutes, 0.020 mL was removed and formulated to 0.24 mg / mL with formulation buffer and stored at 2-8 ° C. After 8 minutes, the remainder of the radiolabel solution was formulated to 0.24 mg / mL and stored at 2-8 ° C. 0.336ml to simulate Friday label⁹⁰The protocol was repeated using Y chloride, 0.323 mL sodium acetate, and 0.333 ml 2B8-MX-DTPA. In both studies, 1 mCi control reaction was performed using the “standard” conditions described above (5 min reaction).
[0088]
c. Added⁹⁰Effect on the amount of Y chloride
On the Monday label, 12mCi⁹⁰Y chloride (0.143 mL) was mixed with 0.143 mL of 50 mM sodium acetate. this⁹⁰The amount of Y is generally used to dose adjust Y2B8⁹⁰20% more than the amount of Y. Bound antibody was added and the sample solution was mixed and then incubated at ambient temperature. The specific activity of the final radiolabel solution was 22.5 mCi / mg antibody. At 2 minutes, 0.020 mL was removed and formulated to 0.24 mg / mL with formulation buffer and stored at 2-8 ° C. After 8 minutes, the remainder of the radiolabel solution was formulated to 0.24 mg / mL and stored at 2-8 ° C. The Friday label is also 0.403ml⁹⁰Performed using Y chloride, 0.403 mL sodium acetate, and 0.333 ml 2B8-MX-DTPA (specific activity was 22.5 mCi / mg antibody). In both studies, 1 mCi control reaction was performed using the “standard” conditions described above (5 min reaction).
[0089]
d. Effects of adding a small amount of antibody conjugate
On the Monday label, 10mCi⁹⁰Y chloride (0.119 mL) and 0.143 mL of 50 mM sodium acetate were mixed. Bound antibody was added 20% less than the amount of antibody normally used (0.267 ml), the sample was mixed and then incubated at ambient temperature. At 2 and 8 minutes, 0.020 mL was removed and the final antibody concentration was formulated to 0.24 mg / mL with formulation buffer and stored at 2-8 ° C. until assayed. The Friday label is also 0.336ml⁹⁰Performed using Y chloride, 0.403 mL sodium acetate, and 0.27 ml conjugate. In both studies, 1 mCi control reaction was performed using the “standard” conditions described above (5 min reaction).
[0090]
e. Effects of combining reagent deviations
In the Monday or Friday label protocol, sodium acetate,⁹⁰The effect when the amount of Y chloride and conjugate was even 20% was simultaneously evaluated. On the Monday label, 12mCi⁹⁰Y chloride (0.143 mL) 0.113 mL of 50 mM sodium acetate, 20% greater⁹⁰Y chloride was mixed with 20% less amount of commonly used sodium acetate. 2B8-MX-DTPA, which represents 20% less than the commonly used antibody, was added and the sample solution was mixed and then incubated at ambient temperature. At 2, 4, 6 and 8 minutes, 0.020 mL was removed and the final antibody concentration was formulated to 0.24 mg / mL with formulation buffer and stored at 2-8 ° C. until assayed. The Friday label is also 0.403ml⁹⁰Performed using Y chloride, 0.387 mL sodium acetate, and 0.267 ml conjugate; 40 μL of sample was removed at the indicated times and formulated with formulation buffer. In both studies, 1 mCi control reaction was performed using the “standard” conditions described above (5 min reaction).
[0091]
2. Measurement of radiation uptake
According to the above assay, the amount of radioactivity bound to the conjugate was measured using a commercially available kit manufactured by Biodex (Tec-Control Radiochromatographic Kit). Using a common micropipettor, 0.5-1 μl of sample was poured onto replicate strips and developed by Biotex instruction incert. The isodata gamma counter was used in a 100-1000 KeV window to measure the radioactivity of half of the strip in a glass tube. Incorporation of radiolabel was assessed by dividing the amount of radioactivity in the upper half of the strip from the total activity determined from both the top and bottom halves. This value was expressed as a percentage and the average value measured.
[0092]
3. Measurement of binding
According to the above protocol, the% binding of the sample to CD20 positive cells was analyzed. However, a negative control sample of HSB cells was not included in these experiments, and SB cells were lyophilized in 5 ml vials instead of microfuge tubes.
[0093]
Essentially, all final Y2B8 samples were diluted 1: 100 with dilution buffer (10.0 μl antibody + 990 μL buffer). The antibody was then diluted again to a concentration in the range of 8 ng / mL by adding 30 μL of 1: 100 dilution to 10 ml of dilution buffer in a 50 mL polypropylene tube.
[0094]
Six to seven vials of lyophilized cells were reconstituted with SWFI and pooled into 50 mL conical tubes. The reconstituted cells (0.5 mL) were aliquoted into three 1.5 mL microfuge tubes and three were tested per sample. Dilution buffer (0.5 mL) was added to three empty microfuge tubes. Diluted antibody (0.5 mL) was added to each tube, tightly capped, mixed and incubated at ambient temperature for 45 min at ambient temperature. After incubation, the cells were pelleted by centrifuging for 5 minutes using a Savant microcentrifuge set at “6” (4000 × g). The supernatant of the sample (0.75 mL) was transferred to a 12 × 75 mm glass tube for measurement of radioactivity using an Isodata gamma counter with an energy window set at 100-1000 KeV.
[0095]
Radioactivity bound to the cells (B) was estimated by subtracting unbound (supernatant) radioactivity from the total radioactivity added. Total radioactivity was measured by measuring the radioactivity of the tube without cells. The% binding was estimated by showing the bound radioactivity as a percentage.
[0096]
To minimize the effect of lot variation between lyophilized cells used to assess binding, binding values were normalized by 1 mCi Y2B8 control prepared using “standard” label conditions. Control samples were prepared for each set of experiments as described earlier in this section.
[0097]
D results
1. Small scale label to predict dose preparation at all scales To confirm that small scale radiolabel response predicts full scale (40 mCi) dose preparation, the radiolabel protocol described above Were used to prepare 1, 3, 10, and 40 mCi Y2B8 doses. These results are shown in Table 6 and show that increasing the scale of the reaction mixture from 1 mCi to 40 mCi has no adverse effect on binding or radiation uptake.
[0098]
[Table 6]

2. Effects of adding a small amount of sodium acetate
When Y2B8 was prepared using 20% less sodium acetate and 60% incubation time, there was substantial binding compared to radiolabels prepared with the following “standard” label conditions: Retained (Table 7 below). Even when the label reaction was performed using Monday label conditions,> 89% of the control binding was retained. Similar results were obtained from Friday dose preparations. Such a shift did not affect radiation uptake regardless of the date the dose was prepared.
[0099]
3.⁹⁰Effect of excessive addition of Y chloride
20% excess⁹⁰When Y2B8 was prepared using Y chloride in combination with 60% longer incubation time than that normally used, it was compared to the radiolabel prepared by the following “standard” label conditions In some cases, substantial binding was retained (Table 7 below). When label response was increased by 8 minutes, binding remained> 90% in either Monday or Friday dose preparations compared to controls. 20% more⁹⁰The addition of Y chloride did not affect radiation uptake regardless of the date the dose was prepared.
[0100]
4. Effects of adding a small amount of antibody conjugate
When Y2B8 was prepared using a 20% lesser amount of conjugate (2B8-MX-DTPA) and with an incubation time of 60% compared to a radiolabel prepared with the following “standard” label conditions: Binding was not significantly affected (Table 7 below). The addition of 20% less conjugate did not affect radiation uptake regardless of the date the dose was prepared.
[0101]
[Table 7]

5. Effects when combined with reagent misalignment
When Y2B8 was prepared using a protocol that caused all four deviations simultaneously, binding was still substantially maintained compared to radiolabeled antibodies prepared with “standard” label conditions (below) Table 8). Even when Monday preparations were incubated 30% longer than the usual maximum of 6 minutes, binding was still> 83%. Regardless of the date the dose was prepared, even after 8 minutes of incubation, radiouptake was not affected by these cumulative deviations.
[0102]
[Table 8]

V. Consideration
In order to reduce the radiation exposed to the operator, a lower label response was evaluated instead of preparing a full scale dose. Thus, we have demonstrated that the 1 mC and 10 mCi labels evaluated in this study predict a 40 mCi preparation at full scale. This result showed that there was no significant difference in binding and radiation uptake above the range of 1 mC to 40 mCi.
[0103]
We have sodium acetate,⁹⁰An error of 20% amount of Y chloride and bound antibody was judged to indicate the possibility of deviation in the radiolabel protocol. Furthermore, incubation for 8 minutes (3 minutes longer than normal) is considered a significant protocol shift. In general,⁹⁰Due to the short half-life of Y chloride, the amount of radioisotope will vary depending on the day of dose preparation. Therefore, the experiments described in this study are intended to present the full range of dose adjustments⁹⁰Y chloride was used at both Monday and Friday concentrations.
[0104]
A dose of Y2B8 prepared using 20% less sodium acetate and 8 minutes incubation retained significant binding (> 89%) compared to standard label conditions. Similar results were obtained from Monday or Friday dose preparations. 20% more on either Monday or Friday⁹⁰Addition of Y chloride and incubation for up to 8 minutes reduced binding compared to standard label conditions. However, the coupling remained> 90% of the standardized emission specification above. Binding was slightly better with Friday dose preparation. Radiation uptake is⁹⁰It was not significantly affected by the increase in the amount of Y chloride.
[0105]
To assess the effects of having all the dose shifts occur at the same time, preparations were made at Monday or Friday doses and compared to incubation times of 2, 4, 6, and 8 minutes. Only when Y2B8 was prepared on Monday using an incubation time of 8 minutes, the standardized specification did not match slightly (83.2% compared to the standardized emission standard of 86.3%).
[0106]
Each reference is incorporated by reference herein.
[Brief description of the drawings]
FIG. 1. A) SB cells washed and prepared using dilution buffer (1 × PBS with 1% bovine serum albumin (BSA), pH 7.2-7.4. 2B8-MX-DTPA lot # 0165A. 90 × 10 at 2 ng / ml Y2B8 for 3 hours to increase cell concentration)⁶Figure when suspended in cells / ml. B) Double reciprocal plot of cell concentration versus bound radioactivity / total radioactivity (B / AT). Immune responsiveness was estimated as 1 / y-intercept × 100. The immunoreactivity and correlation function (R) values were 77.2% and 0.999, respectively.

Claims (20)

A method for radiolabeling a chelator-binding antibody or antibody fragment with ⁹⁰ Y for administration to a patient comprising:
(i) MX-DTPA, phenyl -DTPA, benzyl -DTPA, CHX-DTPA, DOTA, and antibody or antibody fragments bound to a bifunctional chelator selected from the group consisting of a derivative, the ⁹⁰ Y or a salt thereof non Mixing with a solution contained in citrate buffer to make a mixture whose pH is between 3.0 and 6.0 ; and
(ii) ⁹⁰ Y-labeled antibody or antibody fragment may be administered directly to a patient without further purification to separate the radiolabeled antibody or antibody fragment from unincorporated ⁹⁰ Y such, more than 95% radiation uptake, such as at least 5 mCi / mg specific activity, and the radiolabeled antibody or antibody fragment having at least 50% of the binding specificity is made, 25 ° C. from at temperatures of between 43 ° C., incubating the mixture of less than 8 minutes time,
Including methods.   The method of claim 1, wherein the sufficient incubation time is between 2 and 5 minutes.   The method of claim 1, wherein the chelator is MX-DTPA.   The method of claim 1, wherein the acceptable buffer is an acetate buffer. The method of claim 4 , wherein the buffer has a sodium acetate concentration of 10 to 1000 mM.   The method of claim 1, wherein the acceptable buffer comprises a mild radioprotectant. The method of claim 6 , wherein the mild radioprotective agent is ascorbate. _2. The method of claim 1, wherein the antibody fragment is selected from the group consisting of Fab, F (ab ′) ₂ and Fv fragments. The ratio of chelator and the antibody or antibody fragment is between 1 _^1/2 from 1, The method of claim 1.   The method of claim 1, wherein the binding specificity is at least 70%.   2. The method of claim 1, wherein the antibody and antibody fragment are labeled with a specific activity of at least 10 mCi / mg.   2. The method of claim 1, wherein the antibody and antibody fragment are labeled with a specific activity of at least 15 mCi / mg.   2. The method of claim 1, wherein the antibody and antibody fragment are labeled with a specific activity between 5 and 20 mCi / mg.   2. The method of claim 1, wherein the antibody and antibody fragment specifically bind to CD20. 15. The method of claim 14 , wherein the antibody is 2B8. The method of claim 15 , wherein the chelator is MX-DTPA. 17. A method according to claim 16 , wherein the sufficient incubation time is between 2 and 8 minutes. 17. The method of claim 16 , wherein the antibody and antibody fragment are labeled with a specific activity between 5 and 40 mCi / mg. 17. A method according to claim 16 , wherein a level of radiation uptake greater than 96% is achieved. 17. A method according to claim 16 , wherein a level of radiation uptake of up to 99.5% is achieved.

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